Phorbol esters induce changes in mammalian cells in vitro that mimic transformation by oncogenic viruses and chemical carcinogens. One effect caused by these agents is an immediate inhibition of the interaction of EGF with a high-affinity component of binding in intact cells and membranes. These results have raised the possibility that there exist two populations of EGF receptors. It was speculated that the growth-potentiating effects of EGF are directed solely through this high-affinity receptor. Little is known about the underlying mechanisms that regulate EGF receptors. We will study whether heterogeneous EGF binding sites are caused through cooperativity or interconvertible forms of the receptor. Because phorbol esters cannot exert their inhibitory effects on EGF receptors when added directly to membranes, but require prior exposure in intact cells, we will study whether phorbol esters inhibit metabolic processes required for their generation. In particular, the pertinence of tyrosine phosphorylation, protein kinase C, lipid interactions, or limited proteolysis of the receptor on EGF binding will be investigated. Phorbol esters also potentiate the interaction of EGF with intact cells after prolonged exposures, but this is not conserved when membranes are prepared. This result suggests that phorbol esters may direct the intracellular accumulation of EGF into an organelle other than the lysosome, the major site of EGF-receptor degradation. We intend to study the subcellular distribution of EGF after prolonged exposure to phorbol tumor promoters. The possibility of enhanced nuclear translocation of EGF in the presence of phorbol esters has been considered. These approaches will: (1)\provide data relevant to definition of the heterogeneous behavior of EGF receptors on certain cell types; (2)\investigate the molecular mechanisms responsible for immediate phorbol-induced alterations in EGF receptor affinity; and (3)\study the delayed potentiating effects of tumor promoters on EGF receptors. These studies may provide insight into the regulation of mammalian growth control. \(J)